A method of synthesis of a particular polypeptide-chelator described herein, GYK-DTPA (glycyl-tyrosyl-(N-.epsilon.-diethylenetriaminepentaacetic acid)lysine), has been disclosed in U.S. Pat. No. 4,741,900 to Alvarez et al. This synthesis however, depends upon the utilization of the base-cleavable 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group. This strategy has several disadvantages. First, the process is costly due to the use of the expensive Fmoc protecting group and to the labor-intensive preparative HPLC chromatographic purification of end products required to obtain a purified homogeneous product. Secondly, premature loss of the Fmoc group and byproduct formation during scheduled Fmoc removal and DTPA coupling are problematic. The lack of easily purified intermediates necessitates the accumulation of side products, and a preparative high-pressure liquid chromatographic separation is required. In an attempt to address these shortcomings, a new synthesis of GYK-DTPA utilizing the acid-cleavable t-butyloxycarbonyl (Boc) amino protection was devised.
According to U.S. Pat. No. 4,741,900 to Alvarez et al., GYK-DTPA was synthesized as follows: the initial peptide reactant N-Fmoc-glycyl-(O-benzyl-tyrosyl-(.epsilon.-N-carbobenzyloxy) lysine was prepared according to standard solid phase synthetic methods described by Baranz and Merrifield. In The Peptides, Vol. 2, Gross and Meienhoffer (ed.), Acad. press, New York, pp. 3-385, 1980. The derivatized peptide was cleaved from the resin and partially deblocked by bubbling hydrogen bromide (HBr) gas through a suspension of the reaction mixture in trifluoracetic acid containing methoxybenzene (a fifty-fold molar excess over tyrosine) for 60 minutes at room temperature. The resin was removed by filtration and the filtrate evaporated to dryness. The residue was dissolved in a minimum amount of methanol, and the product precipitated with ether and used without further purification.
One mole N-Fmoc-glycyl-tyrosyl-lysine in DMF was reacted with 1 mole DTPA mixed anhydride prepared as described in Krejcarek et al., [Biochem. Biophys. Res. Comm. 77:581-585 (1977)] in which diisopropylethylamine was used for 30 minutes at -15.degree. C., and then maintained at room temperature for 1 hour. The solvent was removed by rotary evaporation and the oily residue dissolved in a small aliquot of DMF. Distilled water was added to precipitate the Fmoc-GYK-DTPA produced. The Fmoc group was removed by addition of an equal volume of 40% dimethylamine in DMF to a solution of Fmoc-GYK-DTPA in DMF, followed by incubation for 30 minutes at room temperature. The solvent was evaporated to dryness and the residue taken up in distilled water. The crude product was purified by extraction with ethyl acetate and the resulting aqueous solution of GYK-DTPA was lyophilized to dryness.
More recently, Alvarez et al., [in Antibody-Mediated Delivery Systems, John D. Rodwell, Ed., Marcel Dekker, Inc., New York, (1988), ch. 10, pp. 306-309] described the synthesis of GYK-DTPA as summarized below. GYK-DTPA was prepared starting with a commercially-available dipeptide, glycyl-tyrosine. The amino terminus of the glycyl-tyrosine starting material was protected by reaction with 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu), to provide the corresponding urethane. Subsequently, a peptide-coupling reaction was performed with N-.epsilon.-benzyloxycarbonyl-lysine methyl ester (H-Lys(.epsilon.-CBZ)-OMe) utilizing the coupling reagents dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (DCC/HOBt). The resulting tripeptide derivative was fully-protected GYK. The urethane protection at the .epsilon.-amino group of the lysine side-chain was cleaved through the agency of hydrobromic acid in acetic acid, providing the Fmoc-GYK-OMe hydrobromide salt, whose amino and carboxy termini remain protected.
The N-Fmoc-GYK(OMe)-HBr and diisopropylethylamine in dry DMF were then reacted with a DTPA mixed anhydride which was prepared by modification of the method of Krejcarek et al. [Biochem. Biophys. Res. Comm. 77: 581-585 (1977)] at about 0.degree. C. for about two hours. The reaction mixture was evaporated and the crude Fmoc-GYK-(OMe)-DTPA solid was dissolved in DMF and 40% dimethylamine in water to remove the Fmoc protecting group. The methyl protecting group was then removed by saponification in 1N NaOH. The final product, GYK-DTPA was isolated using preparative C-18 reverse phase HPLC using a gradient of 0.1% TFA and acetonitrile.
As a result of the Fmoc procedure's multiple problems: high cost, labor-intensive preparative HPLC chromatographic purification of the end product, the potential premature loss of the Fmoc group and byproduct formation during Fmoc removal and DTPA coupling, the lack of easily purified intermediates necessitating the accumulation of side products and the requirement of preparative high-pressure liquid chromatographic separation there remains a need for, a new method for synthesis to remedy and/or avoid these problems.
Other references which describe or relate to o coupling of chelators, including DTPA, to proteins and/or antibodies include the following: U.S. Pat. No. 4,479,930 to Hnatowich et al. describes reaction of an amine of a polypeptide or protein with a dicyclic dianhydride chelator of the formula ##STR1## where R is a linker from 1-25 carbon atoms and which can include nitrogen and/or carboxyl or other groups which do not denature proteins or peptides. The chelators attached to amines include the dicyclic dianhydride of DTPA (see also, Hnatowich, 1982, Int. J. Appl. Radiat. Isot. 33:327-332), U.S. Pat. No. 4,668,503 also to Hnatowich describes labeling of the amine, polypeptide or protein chelator conjugates with Technetium-99 m.
British Patent Application Publication Nos. GB 222579 published Jun. 6, 1990 and GB 224167 published Aug. 28, 1991 describe conjugates of somatastatin peptide analogues and chelating groups including such as DTPA, EDTA, substituted EDTA, DTPA, e.g., p-isothiocyanato-benzyl EDTA or -DTPA, and groups derived from macrocyclic ligands such as 1,4,7,10-tetra-azacyclododecane-N,N',N",N"'-tetra-acetic-D-Phe acid, etc. A particular example includes DTPA-D Phe-Cys-Phe-D-Thr-Lys-Thr-Cys-Thr-OH in which it appears the DTPA is attached to the somatastatin analogue according to a method similar to or the same as the method described by Hnatowich.
Other attempts to develop chelating agents useful for attaching a metal or metal ion to a protein or glycoprotein have included the following. Sundberg et al., 1974, J. Med. Chem. 17:1304 described a complex, multi-step synthesis of 1-(p-benzyldiazonium)-EDTA. EDTA derivatives may not be as useful as DTPA derivatives. Yeh et al., 1979, J. Radioanalytical Chem. 53:327 described a method using 1-(p-carboxymethoxybenzyl)-EDTA which had the same disadvantages as that of Sundberg. Krejcarek et al., 1977, Biochem. Biophys. Res. Comm. 77:581 and more recently Scheinberg et al., 1983, Sci. 215:1511-1513 described attachment of a glycoprotein or protein to a metal ion using a carboxycarbonic anhydride of DTPA. The reaction schemes used by Krejcarek and Scheinberg are not selective for derivatizing a single or particular carboxyl group of the DTPA and can result in undesirable cross-linking when the DTPA derivative is attached to a protein or polypeptide or alternatively can result in undefined DTPA linked products.
It is an object of the present invention to provide a novel synthesis which provides the benefit of using inexpensive starting materials, thereby avoiding the costly use of dipeptides and Fmoc protection reagents.
It is a further object of the present invention to utilize inexpensive reagents and solvents throughout the process. In addition, most of the solvents used in this novel method are volatile and are amenable to solvent recovery, thereby further reducing cost. Another object of the present invention is to minimize the use of DMF which is undesirable.
It is a further object of the present invention to provide stable intermediates which can be easily isolated in excellent purity. The ability to purify these intermediates obviates the need for extensive end-product purification.
In addition it is a further object of the present invention to eliminate the need for a preparative HPLC to purify intermediates or final products.